Several methods have been employed of segregating electrolysis products from analyte. On method employs (i) inserting electrodes into open reservoirs for maximum release of the gaseous products from electrolysis (dihydrogen and dioxygen). Another method employs reservoirs in sufficient volume that the ionic products of electrolysis (protons and hydroxide ion) do not overwhelm the buffering capacity of the buffer solution in the reservoir. However, these methods do not address the issues pertaining to the ionic products of electrolysis.
Techniques such as electrophoresis and chromatography may be used to separate charged molecules such as deoxyribonucleic acid (DNA), ribonucleic acid (RNA) and proteins. Generally, electrophoresis is used to separate charged molecules on the basis of their movement in an electric field. Chromatography on the other hand, is used to separate molecules based on their distribution between a stationary phase and a mobile phase.
Polyacrylamide gel electrophoresis (PAGE) is a standard tool in the study of proteins. Generally, with PAGE, proteins and peptides are exposed to a denaturing detergent such as sodium dodecylsulfate (SDS). SDS binds proteins and peptides. As a result, the proteins/peptides unfold and take on a net negative charge. The negative charge of a given SDS treated protein/peptide is roughly proportional to its mass. An electric field is then applied which causes the negatively charged molecules to migrate through a molecular sieve created by the acrylamide gel. Smaller proteins or peptides migrate through the sieve relatively quickly whereas the largest proteins or peptides are the last to migrate, if at all. Those molecules having a mass between the two extremes will migrate in the gel according to their molecular weight. In this way, proteins that differ in mass by as little as 2% may be distinguished.
Polyacrylamide gel electrophoresis may be used in conjunction with other electrophoretic techniques for additional separation and characterization of proteins. For example, native proteins may be separated electrophoretically on the basis of net intrinsic charge. That is, the intrinsic charge of a protein changes with the pH of the surrounding solution. Thus, for a given protein there is a pH at which it has no net charge. At that pH, the peptide will not migrate in an electric field. Thus, when proteins in a mixture are electrophoresed in a pH gradient, each protein will migrate in the electric field until it reaches the pH at which its net charge is zero. This method of protein separation is known as isoelectric focusing (IEF).
Isoelectric focusing and SDS-PAGE are commonly used in sequence to separate a protein or peptide mixture first in one dimension by IEF and then in a second dimension by PAGE. Isoelectric focusing followed by SDS-PAGE is commonly referred to as 2D-PAGE. Disadvantageously, 2D-PAGE requires the use of bulky equipment. Further, the chemicals required to run 2D-PAGE separations can be expensive and potentially hazardous. Additionally, running 2D-gels can be time consuming and usually requires a skilled technician to obtain satisfactory results. Even then, results may be variable and difficult to reproduce.
Other separation techniques, such as Matrix Assisted Laser Desorption/Ionization Time-Of-Flight Mass Spectrometry (MALDI-TOFMS) are available to separate polar compounds including proteins. However, MALDI-TOFMS requires a substantial investment in expensive equipment and labor, and does not improve upon the core 2D-PAGE separation technology.
Thus, there is a need for improved devices for (i) mitigating the gaseous and ionic products of electrolysis, particularly with regard to microfluidic devices; (ii) a media for electrochromatographic sieving of charged molecules which is easily fabricated and which offers significantly improved performance over conventional sieving media; (iii) a robust, reliable and easily fabricated filter which prevents ambient particles from entering fluidic channels, particularly microfluidic and nanofluidic channels; and (iv) a robust fluidic element which is able to buffer hydraulic pressure surges.